Synthesis of novel sulphamethoxazole derivatives and exploration of their anticancer and antimicrobial properties

A series of new derivatives based on sulfamethoxazole were designed and synthesized in this study. The structures of the new compounds were confirmed based on a comprehensive characterization of spectral data by applied IR and 1H as well as 13C NMR spectroscopy. The prepared compounds were tested for their anticancer and antimicrobial properties. Hydrazone 16b demonstrated convincing anticancer effect against all tested cell cultures such as human prostate carcinoma PPC-1 and human kidney carcinoma CaKi-1 cell lines, and human fibroblasts HF, n = 3. The most promising compound 16b showed higher activity against CaKi-1 cell line than the anticancer drugs axitinib and pazopanib used to treat renal cancer. Also, it was more active in the PPC-1 cell line compared to the approved PARP inhibitor Olaparib. Hydrazone 16b was also found to possess good antimicrobial properties against gram-positive bacteria strains of Staphylococcus aureus, Staphylococcus epidermidis, as well as Bacillus cereus.


Introduction
Synthesis and investigation of heterocyclic compounds have been one of the most important goals of organic and medicinal chemistry due to their broad potential for biological properties. Numerous heterocycle core is rightly considered privileged because as statistics show, more than 85% of all biologically active compounds possess a heterocyclic moiety [1]. Among them, O, N-heterocyclic rings are distinguished not only due to the simple synthesis but also by their wide distribution and bioactivity. Isoxazole derivatives being an entity of this group of compounds make a significant contribution displaying a wide range of important therapeutic properties viz. anti-inflammatory, anti-ulcer [2], anti-cancer [3,4], anti-tubulin [5], antibacterial [6,7], antioxidant [8,9], immunoregulatory [10], antiplatelet [11], antifungal [12], anticonvulsant [13] and many others [14][15][16]. The substitution and modification of isoxazole inert surfaces, stimulating to improve the insertion of compounds as new antibacterial coating agents for medicinal and hospital materials. Sulfonamide-metal complexes have proven to be useful and promising tools in reducing microbial adhesion to inert surfaces and in promoting the development of new antibacterial coatings for medical and hospital materials. Sulfonamide-Au, Cd, Ag, Cu, and Hg complexes demonstrated excellent activity against various microorganisms and confirmed the effectiveness of sulfonamide-metal complexes against rapidly growing mycobacteria (Mycobacterium abscessus, M. fortuitum, and M. massiliense) biofilm [39].
In the light of the above facts and due to the huge development in antimicrobial activities of sulfamethoxazole derivatives, as well as in the continuation of our works in the field of the synthesis and investigation of various sulfonamides [40][41][42][43][44][45] we have synthesized a series of completely new unpublished sulfamethoxazole-based derivatives and have screened them against cancer cell and several standard bacteria and fungus to study their potential in search of novel anticancer and antimicrobial compounds.

Synthesis
Starting materials and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. The reaction course and purity of the synthesized compounds were monitored by TLC using aluminium plates pre-coated with Silica gel with F254 nm (Merck KGaA, Darmstadt, Germany). Melting points were determined with a B-540 melting point analyser (Büchi Corporation, New Castle, DE, USA) and are uncorrected. IR spectra (ν, cm -1 ) were recorded on a Perkin-Elmer Spectrum 100 FT-IR spectrometer (Perkin-Elmer Inc., Waltham, MA, USA) using KBr pellets. NMR spectra were recorded on a Brucker Avance III (400, 101 MHz) spectrometer (Bruker BioSpin AG, Fällanden, Switzerland). Chemical shifts were reported in (δ) ppm relative to tetramethylsilane (TMS) with the residual solvent as internal reference ([D6] DMSO, δ = 2.50 ppm for 1 H and δ = 39.52 ppm for 13 C). Data are reported as follows: chemical shift, multiplicity, coupling constant [Hz], integration and assignment. Mass spectra were obtained on Bruker Daltonic-maXis 4G mass spectrometer (Bruker Daltonics, Bremen, Germany) with ESI ionization. Elemental analyses (C, H, N) were conducted using the Elemental Analyzer CE-440, their results were found to be in good agreement (±0.3%) with the calculated values.
White solid, yield 23 Procedures for the preparation of compounds 5, 6 Method A: A mixture of carboxylic acid 2 (10 mmol, 3.25 g), carbamide (30 mmol, 1.8 g, 5) or potassium thiocyanate (30 mmol, 4.08 g, 6) and acetic acid (25 mL) was heated at reflux for 26 h, then hydrochloric acid was added (to pH 1-2) and the mixture was refluxed for 1 h. After completion of the reaction (TLC), the mixture was cooled down and diluted with water. The formed precipitate was filtered off, washed with water and recrystalized from methanol.
Method B for the synthesis of 6: A mixture of thioureido acid 6a (10 mmol, 3.84 g) and 15% hydrochloric acid (15 mL) was refluxed for 1 h, then cooled down and diluted with water, the formed precipitate was filtered off, washed with water and recrystalized from methanol to give a white solid with the yield of 3.44 g, 94%.
To a mixture of acid 2 (5 mmol, 1.63 g), 4-aminobenzenesulfonamide (6 mmol, 1.03 g) and DMSO (10 mL) triethyl amine (19 mmol, 2.7 mL, dropwise) and HBTU (6.3 mmol, 2.4 g) were added and the mixture was stirred for 20 h at room temperature. After completion of the reaction (TLC) the mixture was diluted with water (20 mL), the formed precipitate was filtered off, washed with water and recrystallized from methanol.
To a solution of hydrazide 4 (1.5 mmol, 0.51 g) in 1,4-dioxane (10 mL) hexane-2,5-dione (4.5 mmol, 0.51 g) and catalytic amount of acetic acid (2 drops) were added and the mixture was heated at reflux for 1 h, the volatile fraction was evaporated under reduced pressure, and the residue was dissolved in methanol, and the obtained solution was diluted with water. The formed precipitate was filtered off, washed with methanol and recrystallized from propan-2-ol.
Light brown solid, yield 0. 18 To a solution of hydrazide 4 (1.5 mmol, 0.51 g) in 1,4-dioxane (10 mL) pentane-2,4-dione (4.5 mmol, 0.45 g) and catalytic amount of hydrochloric acid (1 drop) were added and the mixture was heated at reflux for 1 h, the volatile fraction was evaporated under reduced pressure, and the residue was poured with boiling water, stirred and left to cool down to room temperature. The formed precipitate was filtered off, washed with 2-propanol and recrystallized from propan-2-ol.
Light brown solid, yield 0. 43  To a solution of hydrazide 4 (3 mmol, 1.02 g) in methanol (30 mL) phenyl isocyanate or isothiocyanate (6 mmol) was added dropwise, and the mixture was heated at reflux for 24 h, then volatile fraction was evaporated under reduced pressure, and the residue was dissolved in methanol, and the obtained solution was diluted with water. The formed precipitate was filtered off, washed with methanol and recrystallized from propan-2-ol.   9.04, 9.35, 9.55, 9.85, 9.95, 9. General procedure for the preparation of triazoles 12, 13 A mixture of compounds 10 or 11 (2 mmol) in aqueous 4% sodium solution (10 mL) was heated at reflux for 4 h, then cooled down and acidified with diluted hydrochloric acid (1:1) to pH 2. The formed precipitate was filtered off, washed with water and recrystallized from methanol.

Pharmacology
Cell culturing. The human clear cell renal cell carcinoma line (CaKi-1) was obtained from American Type Culture Collection (ATCC, Manassas, VA). The human prostate carcinoma cell line PPC-1 was originally obtained from ATCC and kindly provided by Prof. Tambet Teesalu (University of Tartu, Estonia). Human foreskin fibroblasts (HF) CRL-4001 were originally obtained from ATCC and kindly provided by Prof. Helder Santos (University of Helsinki, Finland). Cells were cultured in Dulbecco's Modified Eagle's GlutaMAX medium (Gibco (Carlsbad, CA, USA) supplemented with 10,000 U/mL penicillin, 10 mg/mL streptomycin (Gibco), and 10% fetal bovine serum (Gibco). During cell passaging, Tryple Express reagent (Gibco) was used for cell detachment. Cell cultures were grown at 37˚C in a humidified atmosphere containing 5% CO 2 . They were used until the passage of 20.
Compound effect on cancer cell viability. The effect of synthesized compounds on cell viability was studied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich Co., St Louis, MO, USA) assay, as described elsewhere [46]. Briefly, HF, PPC-1, and CaKi-1 cells were seeded in 96-well plates (Corning) in triplicates at a volume of 100 μL at a concentration of 4 × 10 3 cells/well. After 24 h of incubation, the cells were treated with 100 μM of tested compounds. After 72 h, the MTT reagent (at 0.5 mg/mL concentration) has been added to the wells, and cells were incubated for 4 h. Then the medium was aspirated, and the formed purple crystals were dissolved in 100 μL DMSO (Sigma-Aldrich Co.). The absorbance was measured at 570 and 630 nm using a multi-detection microplate reader. The compound effect on cell viability was calculated using a formula (1): Where: A-mean of absorbance of tested compound, A 0 -mean of absorbance of blank (no cells, positive control), A NC -mean of absorbance of negative control (only cells, no treatment). The EC 50 values of the most active compounds 15e, 15k, 16b, and 16d was established by the same MTT procedure. The compound serial dilutions from 100 μM to 3.125 μM have been made in a medium and added to the cells in triplicates. EC 50 value that represent the concentration of a compound causing 50% reduction of cancer cell metabolic activity has been calculated using Hill equation.
Antimicrobial activity by serial dilution in a liquid media. Antibacterial and antifungal susceptibility to the compound 16b was tested using a serial broth dilution technique in Mueller-Hinton broth II (BBL, Cockeysville, USA), as described elsewhere [47]. All procedures were done in aseptic conditions. Standard bacterial and fungal cultures were used: Standard culture of sporic bacteria B. cereus was cultivated for 7 days at 37˚C in Mueller-Hinton II Agar. After sporic bacteria culture had grown, it was washed away from the surface of the broth with sterile physiological solution, and the prepared suspension was being heated for 30 min. at the temperature of 70˚C and diluted till the concentration of spores in 1 ml ranged from 10 x 10 6 to 100 x 10 6 . The fungal culture C. albicans was cultivated for 20-24 hours at 30 ºC in Mueller-Hinton agar. A fungal suspension was prepared in physiological solution according to the turbidity standard 0.5 McFarland.
The compound 16B was dissolved in dimethylsulfoxide right before experiment at a concentration of 20 mM. Then compound serial dilutions from 200 μM to 6.25 μM have been made in a Mueller-Hinton broth and added to the 96-well plates in triplicates at a volume of 50 μl. The prepared microbial cultures were diluted in a Mueller-Hinton brith 150 times and added to the 96-well plate at a volume of 50 μL. In such a way, the final compound concentrations were in the range from 100 μM to 3.125 μM. The wells with only medium served as a positive control, and the wells containing microbial suspension and 0.5% dimethylsulfoxide served as a negative control. The 96-well plates were incubated at 37 ºC for 24 hours (plates with the fungal culture were incubated for 48 hours). Then the absorbance was measured at 600 nm using a multi-detection microplate reader. EC 50 value that represent the concentration of a compound causing 50% reduction of microbial growth has been calculated using Hill equation.
Antimicrobial activity by serial dilution on a solid agar. Antimicrobial activity of compound 16b on solid agar was tested using the same microbial cultures, and the same suspension preparation procedure, as described in above section. All procedures were done in aseptic conditions.
Mueller-Hinton agar was boiled and kept at to 37 ºC during the whole procedure. Compound 16b solution was prepared in DMSO right before experiment. The Agar was mixed with compound solution at different ratios to make the final compound concentrations of 1 mM, 400 μM, 200 μM and 100 μM. Then 10 ml of prepared solution was poured into sterile Petri dishes (85 mm in diameter) and kept in aseptic conditions while the agar solidified. The agar with 0.5% of DMSO served as a negative control. With the help of loop standard microbial cultures by 0.1 ml were poured on the surface of prepared agar with investigative compound. Then Petri dishes were kept for 24 hours in thermostatic conditions.
The minimal dilution, i.e. the lowest concentration in μg/mL of the investigative and comparison compounds that inhibited visible growth of microorganisms was the minimal inhibitory concentration (MIC).
Statistical analysis. All biological experiments were repeated at least three times, calculating the mean and standard deviation. The data was processes using Microsoft Office Excel 365 software (Microsoft Corporation, Redmond, WA, USA). Statistical analysis was performed by using Student's t-test. The level of significance was set as p < 0.05.

Chemistry
In this study, sulfamethoxazole was used as initial compound. To obtain carboxylic acid 2 (Scheme 1) sulfamethoxazole 1 was reacted with acrylic acid in water. The refluxing of the reaction mixture for 24 hours yielded compound 2, which then was applied for the preparation of In the route to acid hydrazide 4 ester 3 was prepared and used as a building block in the reaction with hydrazine monohydrate. A viscous product 4 was separated from the reaction mixture after heating at reflux in propan-2-ol for 24 hours.
The interaction of β-alanine 2 with urea or potassium thiocyanate in refluxing acetic acid followed by the acidification of the reaction mixture with hydrochloric acid to cyclize the intermediate ureido or thioureido acid resulted in 1-aryl-5,6-dihydrouracil 5 and its 2-thio analogue 6, respectively. In the 1 H NMR spectra, the additional peaks at 11.45 ppm (5) and 11.55 ppm (6) were assigned to the protons of NH groups of the cyclic fragment. The 13 C NMR spectrum of compound 5 showed resonances at 157.48 and 170.42 ppm, which were attributed to 2C = O of the formed cycle, as well as in the spectrum of compound 6 spectral lines at 170.48 and 179.62 (6) ppm confirmed a ring bearing the carbonyl and thiocarbonyl groups. To isolate intermediate 6a a solution of thioxotetrahydropyrimidinone 6 in 5% sodium hydroxide was stirred at room temperature for 24 hours then filtered off and the filtrate was acidified with glacial acetic acid to pH 6-7. The comparison of the 1 H NMR spectra of 6 and 6a demonstrated the characteristic shifts of the protons' triplets of the NCH 2 CH 2 fragment from 2.81 and 3.94 ppm for 6 to 2.54 and 4.14 ppm for 6a. Also, the OH hydrogen appearing at 12.24 ppm and broad singlet of the NH 2 protons in the aromatic area at 7.22 ppm in the 1 H NMR spectrum of thioureido acid 6a confirmed the obtained structure.
The formation of amide 7 was performed by coupling carboxylic acid 2 and sulfanilamide with HBTU as coupling agent and TEA in dimethyl sulfoxide for 20 hours at room temperature. The dilution of the reaction mixture with water afforded the target compound 7. The 1 H and 13 C NMR spectra of compound 7 are in excellent agreement with the predicted ones.
In the NMR spectra of compound 8 the singlets at 5. Semicarbazide 10 and thiosemicarbazide 11 were prepared upon refluxing compound 4 with phenyl isocyanate or isothiocyanate in methanol. Afterwards, they were cyclized to triazoles 12 and 13 by refluxing in aqueous 4% sodium hydroxide solution for 4 hours and subsequent acidification of the reaction mixture with diluted hydrochloric acid to pH 2. Comparison of the NMR spectra of compounds 10, 11 and 12, 13 demonstrated obvious characteristic differences, which approve the target structures.
In the 1 H NMR spectrum of semicarbazide 10, singlets at 8.03, 8.36, 8.70, 8.90, 9.03 and a sharp single peak at 9.74 ppm were observed due to the presence of a CONHNHCONH moiety protons. The same spectrum of its thio analogue 11 displayed peaks at 9.04, 9.35, 9.55, 9.85, 9.95 and 9.99 ppm integrated for 3 protons which confirm the presence of a CONHNHCSNH fragment. Analysing the 1 H NMR spectra of their cyclic derivatives 12 and 13, intense peaks in the down-field of the spectra (11.73 ppm for 12 and 13.77 ppm for 13) were observed. They were attributed to the NH of the triazole ring. The 13 C NMR spectra of compounds 10-13 showed resonances in agreement with target structures.
When acid hydrazide 4 was treated with isatin in the equimolar amount in 1,4-dioxane in the presence of a catalyst glacial acetic acid at 100˚C for 3 hours the hydrazone 14 possessing the 2-oxindole moiety was obtained. The success of the reaction was confirmed by the data of spectroscopy and the elemental analysis. In the 1 H NMR spectrum, the double sets of the peaks of the NHCO proton arise at 10.68, 10.79 and 12.57, 12.93 ppm with the intense ratio of 25:75, indicating the presence of a mixture of the Z/E isomers in DMSO-d 6 solution, where usually the Z-form predominates [48,49]. In the 1 H NMR spectrum of 14 the resonance of the Z isomer appeared at a lower-field area of the spectrum with the respect of the peak of the E-form [50]. The 13 C NMR spectrum exhibited the characteristic resonances belonging to the carbons of the 2-oxindole and amide carbonyls, which appeared at 162.49 and 169.91 ppm.
Hydrazones 15a-k, as well as 16a-d, were prepared by condensing of hydrazide 4 with the corresponding aromatic aldehyde (for 15) or carbaldehyde (for 16) in refluxing 1,4-dioxane with the addition of hydrochloric acid as a catalyst. To obtain hydrazone 15k with the 1-naphthalenyl moiety in the molecule catalytic amount of glacial acetic acid was used instead of HCl. The 1 H NMR spectra of the target hydrazones showed a mixture of two isomers in DMSO-d 6 solutions which form due to the presence of an amide bond in the structures and the restricted rotation around this bond. The resulting hydrazones exist as Z and E-amide conformers with an intensity ratio found to be 60:40 with the Z-conformer predominating [48,51] and arising up-field for almost all hydrazones except 16c based on NH signal integration. In the 1 H NMR spectrum of compound 16c with the thiophene substituent in the molecule, only one intense peak for NH proton at 11.34 ppm was found. Comparison of the 1 H NMR spectra of hydrazide 4 with hydrazones 15 and 16 showed a substantial difference between the chemical shifts of their NH signals. In the spectra of 15 and 16 double sets of the amide NH proton signals were visible in the range of 11.13-11.88 ppm while the hydrazide NH proton signal appeared at 9.03 ppm. In addition, the aromatic region of the spectra revealed an increase in the peaks integrated for the corresponding number of protons of the newly attached aromatic ring.
After confirming the structures of the resulting compounds, they were used for the evaluation of their anticancer and antimicrobial properties.

Pharmacology
Anticancer activity. Tested compounds demonstrated different activity against human prostate carcinoma PPC-1 and kidney carcinoma CaKi-1 cell lines (Fig 2). The cytotoxicity of sulfamethoxazole derivatives against both types of cancer cells was similar. CaKi-1 cells were slightly more sensitive to several compounds, especially 14, 15a, 15d, 15f, 15g, 15h, 15j, and 15k containing phenyl, 4-fluorophenyl-, 4-chlorophenyl-, 4-methoxyphenyl-, 3-and 4-nitrophenyl as well as 1-naphthyl substituents in the structure. In general, compound selectivity towards cancer cell lines was not high. The most active compound was 16b, and it showed practically the same effect on the viability of all tested cell cultures (Fig 2). The result was expected due to the presence of a 5-nitrofuryl moiety in the hydrazone molecule, as such type of compounds are characterized by the pronounced anticancer activity [52].
For further experiments four most promising compounds were selected, that reduced cell viability up to about 40% and more, namely, compounds 15e, 15k, 16b, and 16d.
Compound 16b was confirmed to be the most active one of all tested sulfamethoxazole derivatives (Fig 3), however, it was not selective towards cancer cells (EC 50 = 10.4 ± 3.0 μM against PPC-1, EC 50 = 12.4 ± 1.6 μM against CaKi-1, and EC 50 = 9.5 ± 2.7 μM against HF). The least active and selective was compound 15k (EC 50 = 181.0 ± 8.5 μM against PPC-1, EC 50 = 103.5 ± 6.5 μM against CaKi-1, and EC 50 = 201.5 ± 5.6 μM against HF). The compound 15e showed a similar effect on CaKi-1 and HF cell viability, while activity against PPC-1 was even lower. However, the activity of compounds in cell monolayer may not reflect the activity in vivo [53], thus their effects on three-dimensional models would be worthy to study, taking into account that such systems better represent tumour microenvironment and its properties [54].
Metastatic renal cell carcinoma is a very aggressive cancer type, and the 5-year survival rate of the patients is less than 12% [55]. Tyrosine kinase inhibitors, such as sorafenib, are approved to treat this cancer, but in most of patients these drugs become not effective due to the development of cancer resistance to the treatment [56]. The activity of sunitinib against CaKi-1 cells is higher (EC 50 = 2.2 μM) [57] compared to the most active tested sulfamethoxazole derivative in our studies. On the other hand, two anticancer compounds that are also approved for the treatment of renal cancer, axitinib and pazopanib, possess lower activity (EC 50 is 25 and 30 μM after 72 hours of incubation, accordingly) [58]. Thus, the activity of most active identified compounds from tested sulfamethoxazole derivatives is comparable, and they could be modified and further developed for this type of cancer.
Despite relatively good response to the available treatment, prostate cancer is the fifth leading cause of death worldwide [59]. It affects usually older population of men, and the treatment could be problematic. There is a need for newer, more effective agents [60]. The most active compound 16b shows even higher activity in PPC-1 cell line than the approved anticancer drug, PARP inhibitor, Olaparib (EC 50 values in prostate cancer cell lines are in range from 5.4 to even 73.2 μM) [61].
In summary, synthesized sulfamethoxazole derivatives show relatively moderate cytotoxic effect on prostate and kidney carcinoma cell lines and are not very selective. The most active candidate bearing a 5-nitrofuryl fragment shows a promising activity against both cancer cell lines. However, it could be mentioned, that 2-chlorophenyl, naphthalen-1-yl and 5-nitrothiophen-2-yl moieties are also worthy of attention and could be further used for searching and developing more selective compounds against both types of cancer.
Antimicrobial activity. Compound 16b showed relatively high activity in cancer cell lines, but it was not very selective to them compared to fibroblasts. Sulfamethoxazole is widely used as antimicrobial medicinal substance, thus we decided to explore the effects of this compound against different microbial cultures.
Similar results were obtained from experiments on solid agar ( Table 1). The MICs values were higher compared to the EC 50 values, and this observation is consistent with those made but other researchers [62].
Activity of compound 16b was higher against S. aureus compared to its original structural component-sulfamethoxazole, which is still used nowadays in combination with  trimethoprim [63]. Considering the fact that most gram-negative microorganisms are not sensitive to sulfamethoxazole, it could be possible to explain the selectivity of 16b towards grampositive bacteria [64]. Incorporation of 5-nitro-2-furyl fragment in its structure did not grant the activity against gram-negative rods, despite this structural component is present in nitrofurans and is responsible for their mechanism of action [65]. In summary, 16b compound could be a potential drug-lead molecule for the new antimicrobial agents, especially those acting on drug resistant bacteria.

Conclusions
A series of 3-((4-(N-(5-methylisoxazol-3-yl)sulfamoyl)phenyl)amino)propanoic acid and its hydrazide derivatives were synthesized and evaluated for their anticancer and antimicrobial activity. Hydrazone with a 5-nitrofuryl moiety in the structure was found to show the best efficiency on the viability of tested cancer cell cultures, i.e. human prostate carcinoma PPC-1 and human kidney carcinoma CaKi-1 cell lines. The results of an antimicrobial test in a liquid medium demonstrated that gram-positive bacteria strains of S. aureus and S. epidermidis and B. cereus were the most sensitive to its action, while gram-negative bacteria strains of E. coli, K. pneumoniae, P. aeruginosa, and P. mirabilis showed complete resistance for compound.